|M.Sc Student||Kanteev Margarita|
|Subject||Structure-Function Investigation of a Manganese|
The Role of the Disulfide Bond in the
|Department||Department of Chemistry||Supervisor||Professor Noam Adir|
|Full Thesis text|
In the cyanobacterium Synechocystis sp. PCC6803, high affinity manganese import is carried out by an ABC transporter. This system contains the periplasmic substrate-binding membrane-anchored lipoprotein component (MntC), which is important for the biogenesis of Photosystem II.
The structure of the MntC protein from Synechocystis sp. PCC 6803 has been determined using X-ray crystallography to a resolution of 2.9Å. The protein was crystallized as a trimer in the asymmetric unit. The most important difference between the MntC and previously determined solute binding proteins is the presence of a disulfide bond between Cys219 and Cys268 near the metal binding site. It was found that due to reduction of the disulfide bond, protein affinity towards the Mn+2 is decreased.
Our goals in the study presented in this thesis were: 1) improve the resolution of the MntC structure to afford structural analysis of greater precision and 2) to try and better understand the role of the disulfide bond.
The MntC gene was recloned into a new vector which allows an attachment of 6xHis tag, allowing purification of soluble protein by chelation - chromatography.
The 6xHis tag improved the purification step, but it was found to disturb the formation of the crystal lattice and crystals diffract to a resolution lower than 3.5Å. However, the structure of the MntC protein was solved to a 4.5Å resolution, and the metal ion was found in the binding site of the protein. In order to determine the identity of the metal ion, X-ray fluorescence of a single crystal was performed, and the anomalous data was collected at 0.976 Å. The results shows that the MntC can bind Zn2+ in vitro, even though it has a much lower affinity to Zn2+ as compared to Mn2+.
In order to check the role of the disulfide bond, site directed mutagenesis was performed. The mutated proteins were isolated but could not be crystallized due to protein degradation. We found that the mutated proteins degrade either due to enhanced sensitivity to proteases or, and perhaps surprisingly, due to the creation of a novel proteolytic activity within the protein itself.
In order to compare the Mn2+ affinity of the native MntC protein versus that of the mutated MntC, ICP-MS analyses were performed. As was expected, the mutated protein without the disulfide bond and the native protein with reduced disulfide bond have lower affinity to the Mn+2 ions.